1. Field of the Invention
The invention relates to mediation of specific cytokine induction. More particularly, the invention relates to modulating specific cytokine expression in vivo.
2. Summary of the Related Art
Cell-mediated immunity (CMI) is an important mechanism for host defense against a broad range of infectious diseases. CMI is largely controlled through expression of specific cytokines. Le and Vilcek, Laboratory Investigation 61: 588-602 (1989) teaches that the cytokine IL-6, produced mostly by B-lymphocytes, monocytes and Th2 cells, promotes release of acute phase reactants and contributes to T cell activation. Trinchieri et al., Ann. Rev. Immunol. 13: 252-276 (1995) and Res. Immunol. 146: 419-656 (1995) disclose that the recently discovered cytokine IL-12 is critical to the initiation of CMI.
IL-12 most likely acts first upon resting natural killer (NK) cells, which express the IL-12 receptor. Gazzinelli et al., Proc. Natl. Acad. Sci. USA 90: 6115-6119 (1993) teaches that upon stimulation with IL-12, NK cells produce high levels of IFN-gamma, which is a potent stimulator of macrophage effector functions against invasive pathogens, and which further enhances IL-12 synthesis by macrophages previously triggered by an infectious agent.
At another level, IL-12 appears to act by driving the differentiation of T helper cell precursors (Thp) toward production of Th1 cells, which produce IL-2, IFN-gamma and TNF-beta, thereby driving the CMI response. Seder et al., teaches that this process begins by recognition of specific antigen by Thp, which produces IL-2 and subsequently IL-12 receptor. The Thp cell then differentiates into a Th1 cell if IL-12 is present, or into a Th2 cell in an IL-4 environment. Gazzinelli et al., J. Immunol. 153: 2533-2543 (1994) teaches that the Th1 cells, once differentiated, do not require IL-12 as a co-stimulatory molecule to produce cytokines and mediate resistance against pathogens.
Miller and Krangel, Crit. Rev. Immunol. 12: 17-46 (1992) and Taub et al., J. Clin. Invest. 95: 1370-1376 (1995) teach that chemokines are one superfamily of cytokines that play important roles in recruitment and activation of lymphocytes to sites of inflammation. Gallo et al., Science 274: 1393-1395 (1996) teaches that certain chemokines, such as RANTES, MIP-1xcex1 and MIP-1xcex2 can suppress the replication of macrophage-tropic HIV strains in infected T cell cultures, and therefore may play important roles in the regulation of virus replication and are potentially useful as anti-viral therapeutics.
During bacterial infections, the natural immune response is characterized by the production of various cytokines which are involved in CMI. Uyttenhove et al., J. Exp. Med. 167: 1417-1427 (1988) teaches that IL-6 production is stimulated by bacterial infection. Murray, Diagn. Microbiol. Infect. Dis. 13: 411-421 (1990) teaches that IFN-gamma is produced in response to bacterial infection. Trinchieri, Blood 84: 4008-4027 (1994) discloses IL-12 induction resulting from bacterial infection.
The bacterial components responsible for such cytokine induction have recently been investigated. Yamamoto et al., Microbiol. Immunol. 36: 983-997 (1992) showed that bacterial DNA, but not mammalian DNA, boosts lytic activity of NK cells as well as IFN-gamma production, and proposed that this effect was caused by palindromic sequences present in bacterial DNA. More recently, Klinman et al., Proc. Natl. Acad. Sci. USA 93: 2879-2883 (1996) discloses that the ex vivo induction of IL-6, IL-12 and IFN-gamma by bacterial DNA is mediated by a structural motif of an unmethylated CpG dinucleotide preceded by two purines and followed by two pyrimidines and that this effect can be duplicated by an oligonucleotide of at least eight nucleotides containing such a structural motif. These authors noted that such effects may confound studies involving antisense, gene therapy, or plasmid DNA vaccines produced in bacteria.
Interest in developing drugs modeled from microbial products that induce CMI has been expressed. Gazzinelli, Molecular Medicine Today, June 1996, pp. 258-267, notes that such compounds would have a broad application in immunotherapy. However, Gazzinelli also teaches that uncontrolled IL-12 synthesis may cause excessive activation of the immune system, resulting in severe host tissue damage and death. Gazzinelli concludes such toxicity is likely to limit the use of IL-12 therapy in humans.
There is, therefore, a need for new approaches to modulating specific CMI-inducing cytokines in vivo. Such approaches should stimulate production of the desired cytokines without substantially inducing undesired cytokines and without causing unwanted toxic side effects. Ideally, such approaches should protect against infection by a pathogenic agent or against tumor development.
The invention provides new methods for modulating specific CMI-inducing cytokines in vivo. Such new approaches result in stimulation of the cytokines IL-6, IL-12 and IFN-gamma and chemokines (MIP-1xcex1 and MIP-1xcex2) without substantially inducing undesired cytokines. Moreover, the methods according to the invention provide protection against infection by pathogenic agents or against tumor development.
In a first aspect, the invention provides a method for elevating levels of IL-12 in a mammal, including a human. This method according to the invention comprises measuring a baseline level of IL-12 in the mammal, administering to the mammal an oligonucleotide having a structural motif which induces IL-12 expression in vivo, and measuring the level of IL-12 in the mammal after such administration, wherein the level of IL-12 measured after such administration is higher than the level of IL-12 measured before such administration. In a second aspect, the invention provides a method for elevating expression of IL-12 mRNA in a mammal, including a human. This method according to the invention comprises measuring a baseline level of IL-12 mRNA in cells from the mammal, administering to the mammal an oligonucleotide having a structural motif which induces IL-12 expression in vivo, and measuring the level of IL-12 mRNA in cells from the mammal after such administration, wherein the level of IL-12 mRNA measured after such administration is higher than the level of IL-12 mRNA measured before such administration. In a third aspect, the invention provides a method for prophylactically protecting a mammal, including a human, from infection by a pathogen. In the method according to this aspect of the invention, an oligonucleotide having a structural motif which induces IL-12 expression in vivo is administered to a mammal which is not expressing symptoms of infection by the pathogen. The oligonucleotide is administered in an amount and for a time sufficient to prevent successful infection by the pathogen. In a fourth aspect, the invention provides a method for therapeutically treating a mammal, including a human, which is infected by a pathogen. In the method according to this aspect of the invention, an oligonucleotide having a structural motif which induces IL-12 expression in vivo is administered to a mammal which is infected by the pathogen. The oligonucleotide is administered in an amount and for a time sufficient to eliminate or reduce symptoms of infection by the pathogen. In a fifth aspect, the invention provides a method for reducing tumor growth in a mammal, including a human, which has a tumor. In the method according to this aspect of the invention, an oligonucleotide having a structural motif which induces IL-12 expression in vivo is administered to a mammal which has a tumor. The oligonucleotide is administered in an amount and for a time sufficient to eliminate or reduce tumor growth in the mammal.
In the methods according to each aspect of the invention, the mammal to which the oligonucleotide may be administered includes humans. Further, in the methods according to each aspect of the invention, the oligonucleotides administered to the mammals may take the form of particular preferred embodiments. In one preferred embodiment, the oligonucleotide has the nucleotide sequence Nn1-Nn2-CpG-Nn3-Nn4, wherein N represents any nucleoside, n1 and n4 each independently represent a number from 0 to 50, n2 represents a number from 0 to 50 and n3 represents a number from 0 to 50 such that n2+n3 equals from about 6 to about 100, wherein the underlined region represents a nucleoside phosphodiester or phosphorothioate region or a mixed backbone region having phosphodiester and phosphorothioate nucleosides, wherein CpG represents a cytosine-guanosine dinucleoside phosphorothioate or phosphodiester dinucleoside, wherein the cytosine has a cytidine base having an unmethylated 5-position, and wherein at least one of n1, n2, n3, and n4 comprises four consecutive guanosine nucleosides.
The invention relates to in vivo mediation of specific cytokine induction. The patents and publications identified in this specification are within the knowledge of those skilled in this field and are hereby incorporated by reference in their entirety.
The invention provides new methods for modulating specific CMI-inducing cytokines and chemokines in vivo. Such new approaches result in stimulation of the cytokines IL-6, IL-12 and IFN-gamma and chemokines (MIP-1 xcex1and MIP-1 1xcex2) without substantially inducing undesired cytokines. Moreover, the methods according to the invention provide protection against infection by pathogenic agents or against tumor development.
In a first aspect, the invention provides a method for elevating levels of IL-12 in a mammal, including a human. This method according to the invention comprises measuring a baseline level of IL-12 in the mammal, administering to the mammal an oligonucleotide having a structural motif which induces IL-12 expression in vivo, and measuring the level of IL-12 in the mammal after such administration, wherein the level of IL-12 measured after such administration is higher than the level of IL-12 measured before such administration. Preferably, the level of IL-12 is measured in serum and reference is made to serum IL-12 levels or serum IL-12 protein levels. In one preferred embodiment, the levels of IL-12 are measured using ELISA analysis, with antibodies specific for IL-12 protein. However, those skilled in the art will recognize that numerous methods for determining specific protein concentrations are known in the art, and that any of these methods may be used, including without limitation radioimmunoassay, quantitative immunoprecipitation, radial immunodiffusion and cell-based functional assays for IL-12 activity. Preferably, the levels of IL-12 are measured from about 1 minute to about 1 day before administration of the oligonucleotides, to minimize the likelihood of intervening infection, which could alter IL-12 baseline levels. It is preferred that the levels of IL-12 be measured again from about eight hours to about 4 days after oligonucleotide administration, and most preferably about 1 day after oligonucleotide administration. In a preferred embodiment, the method according to this aspect according to the invention also results in increased levels of IL-6 and/or IFN-gamma, either or both of which can be conveniently measured as described above for IL-12. In particularly preferred embodiments, post-administration IL-6 and/or IFN-gamma levels are measured from about one day to about one week after administration of the oligonucleotide, and most preferably about three days after administration of the oligonucleotide.
In a second aspect, the invention provides a method for elevating expression of IL-12 mRNA in a mammal, including a human. This method according to the invention comprises measuring a baseline level of IL-12 mRNA in cells from the mammal, administering to the mammal an oligonucleotide having a structural motif which induces IL-12 expression in vivo, and measuring the level of IL-12 mRNA in cells from the mammal after such administration, wherein the level of IL-12 mRNA measured after such administration is higher than the level of IL-12 mRNA measured before such administration. Preferably, the level of IL-12 mRNA is measured in lymphoid cells, and most preferably in extracts of lymphoid cells. In one preferred embodiment, the lymphoid cells are peripheral blood lymphocytes. In a preferred embodiment, the levels of IL-12 mRNA are measured using RNAse protection assay analysis, with primers and probes specific for IL-12 mRNA. However, those skilled in the art will recognize that numerous methods for determining specific mRNA concentrations are known in the art, and that any of these methods may be used, including without limitation dot blotting, slot blotting, Northern blotting, in situ hybridization, and in vitro translation coupled with cell-based functional assays for IL-12 activity. Preferably, the levels of IL-12 mRNA are measured from about 1 minute to about 1 day before administration of the oligonucleotides, to minimize the likelihood of intervening infection, which could alter IL-12 mRNA baseline levels. It is preferred that the levels of IL-12 be measured again from about four hours to about 2 days after oligonucleotide administration, and most preferably from about four hours to about 1 day after oligonucleotide administration. Preferably, the peak levels of IL-12 mRNA should exceed the baseline levels by about 10-fold or more. In a preferred embodiment, the method according to this aspect according to the invention also results in increased levels of IL-6 mRNA, which can be conveniently measured as described above for IL-12 mRNA. In particularly preferred embodiments, post-administration IL-6 mRNA levels are measured from about four hours to about 1 day after administration of the oligonucleotide. Most preferably peak levels of IL-6 mRNA should exceed baseline levels by about 8-fold or more.
In a third aspect, the invention provides a method for prophylactically protecting a mammal, including a human, from infection by a pathogen. In the method according to this aspect of the invention, an oligonucleotide having a structural motif which induces IL-12 expression in vivo is administered to a mammal which is not expressing symptoms of infection by the pathogen. The oligonucleotide is administered in an amount and for a time sufficient to prevent successful infection by the pathogen. Preferably, such administration should be parenteral, oral, sublingual, transdermal, topical, intranasal or intrarectal. Preferably, the oligonucleotide should be formulated in a physiologically acceptable carrier or diluent, including without limitation saline and/or an adjuvant. Administration of the oligonucleotides can be carried out using known procedures at dosages and for periods of time effective to prevent symptoms or surrogate markers of the disease from appearing. When administered systemically, the oligonucleotide is preferably administered at a sufficient dosage to attain a blood level of oligonucleotide from about 0.01 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of oligonucleotide will range from about 0.1 mg oligonucleotide per patient per day to about 200 mg oligonucleotide per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more IL-12 inducing oligonucleotide to an individual as a single treatment episode. In a preferred embodiment, after the oligonucleotide is administered, one or more measurement is taken of levels of IL-12 protein or mRNA, to assess the effectiveness of the prophylaxis.
In a fourth aspect, the invention provides a method for therapeutically treating a mammal, including a human, which is infected by a pathogen. In the method according to this aspect of the invention, an oligonucleotide having a structural motif which induces IL-12 expression in vivo is administered to a mammal which is infected by the pathogen. Preferably, the oligonucleotide is administered as soon as possible after symptoms are first observed, and most preferably within one day to one week from the time at which symptoms are first observed. The oligonucleotide is administered in an amount and for a time sufficient to eliminate or reduce symptoms of infection by the pathogen. Preferably, such administration should be parenteral, oral, sublingual, transdermal, topical, intranasal or intrarectal. Preferably, the oligonucleotide should be formulated in a physiologically acceptable carrier or diluent, including without limitation saline and/or an adjuvant. Administration of the oligonucleotides can be carried out using known procedures at dosages and for periods of time effective to reduce or eliminate symptoms or surrogate markers of the disease. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more IL-12 inducing oligonucleotide to an individual as a single treatment episode. In a preferred embodiment, after the oligonucleotide is administered, one or more measurement is taken of levels of IL-12 protein or mRNA, to assess the effectiveness of the intervention.
In a fifth aspect, the invention provides a method for reducing tumor growth in a mammal, including a human, which has a tumor. In the method according to this aspect of the invention, an oligonucleotide having a structural motif which induces IL-12 expression in vivo is administered to a mammal which has a tumor. The oligonucleotide is administered in an amount and for a time sufficient to eliminate or reduce tumor growth in the mammal. Preferably, the oligonucleotide is administered as soon as possible after the tumor is first detected. The oligonucleotide is administered in an amount and for a time sufficient to eliminate or reduce symptoms of infection by the pathogen. Preferably, such administration should be parenteral, oral, sublingual, transdermal, topical, intranasal or intrarectal. Preferably, the oligonucleotide should be formulated in a physiologically acceptable carrier or diluent, including without limitation saline and/or an adjuvant. Administration of the oligonucleotides can be carried out using known procedures at dosages and for periods of time effective to reduce or eliminate symptoms or surrogate markers of the disease. When administered systemically, the oligonucleotide is preferably administered at a sufficient dosage to attain a blood level of oligonucleotide from about 0.01 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of oligonucleotide will range from about 0.1 mg oligonucleotide per patient per day to about 200 mg oligonucleotide per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more IL-12 inducing oligonucleotide to an individual as a single treatment episode. In a preferred embodiment, after the oligonucleotide is administered, one or more measurement is taken of levels of IL-12 protein or mRNA, to assess the effectiveness of the intervention.
In the methods according to each aspect of the invention, the mammal to which the oligonucleotide may be administered includes humans. Further, in the methods according to each aspect of the invention, the oligonucleotides administered to the animals may take the form of particular preferred embodiments. In one preferred embodiment of the methods according to each aspect of the invention, the oligonucleotide has the nucleotide sequence Nn1-Nn2-CpG-Nn3Nn4, wherein N represents any nucleoside, n1 and n4 each independently represent a number from 0 to 50, n2 represents a number from 0 to 50 and n3 represents a number from 0 to 50 such that n2+n3 equals from about 6 to about 100, wherein the underlined region represents a nucleoside phosphodiester or phosphorothioate region or a mixed backbone region having phosphodiester and phosphorothioate nucleosides, wherein CpG represents a cytosine-guanosine dinucleoside phosphorothioate or phosphodiester dinucleoside, wherein the cytosine has a cytidine base having an unmethylated 5-position, and wherein at least one of n1, n2, n3, and n4 comprises four consecutive guanosine nucleosides. Most preferably, n1 and n4 each independently represent a number from 0 to 10, n2 represents a number from 0 to 20 and n3 represents a number from 0 to 20 such that n2+n3 equals from about 6 to about 40.
In another preferred embodiment, the oligonucleotide has the nucleotide sequence Nn1-Nn2-Pu-Pu-CpG-Py-Py-Nn3-Nn4, wherein N represents any nucleoside, Pu represents a purine, Py represents a pyrimidine, n1 and n4 each independently represent a number from 0 to 50, n2 represents a number from 0 to 50 and n3 represents a number from 0 to 50 such that n2+n3 equals from about 2 to about 100, wherein the underlined region represents a nucleoside phosphodiester or phosphorothioate region or a mixed backbone region having phosphodiester and phosphorothioate nucleosides, and wherein CpG represents a cytosine-guanosine dinucleoside phosphorothioate or phosphodiester dinucleoside, wherein the cytosine has a cytidine base having an unmethylated 5-position. Most preferably, n1 and n4 each independently represent a number from 0 to 10, n2 represents a number from 0 to 20 and n3 represents a number from 0 to 20 such that n2+n3 equals from about 2 to about 40.
In another preferred embodiment, the region(s) of the oligonucleotide outside the underlined region, when present, may optionally include polymers of two or more deoxyribonucleotide, ribonucleotide, or 2xe2x80x2-O-substituted ribonucleotide monomers, or any combination thereof. Such polymers may be internucleosidically linked by any internucleoside linkage, including without limitation, phosphodiester, phosphotriester, phosphorothioate, phosphorodithioate, phosphoramidate, alkylphosphonate, carbamate, and amide (PNA) linkages, or any combination of the same. Those skilled in the art will recognize that the type of internucleoside linkages in such region(s) is not critical. For purposes of the invention the term xe2x80x9c2xe2x80x2-O-substitutedxe2x80x9d means substitution of the 2xe2x80x2 position of the pentose moiety with an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group, but not with a 2xe2x80x2-H group. Such region(s) also encompass such polymers having chemically modified bases or sugars and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane.
The methods according to the invention are useful for preventing or treating pathogenic infections in animals, such as in mice. In addition, the methods according to the invention are useful as prophylactic and therapeutic approaches to pathogenic infections in humans. Such pathogens include numerous pathogenic viruses. Preferred viruses include without limitation human immunodeficiency virus (type 1 or 2), influenza virus, herpes simplex virus (type 1 or 2), Epstein-Barr virus, human and murine cytomegalovirus, respiratory syncytial virus, hepatitis B virus, hepatitis C virus and papilloma virus. Preferred pathogens also include eukaryotic or prokaryotic pathogens, including without limitation, Plasmodium falciparum, Plasmodium malarie, Plasmodium ovale, Schistosoma spp., Streptococcus spp., Staphylococcus spp., Pneumococcus spp., Neisseria spp., Vibrio spp., E. coli and Mycobacterium tuberculosis.